Direct detection of biomolecules in polyacrylamide gel

ABSTRACT

An improved method for detecting a target biomolecule directly in a polyacrylamide gel in which it has been separated from other substances. The improvement resides in, prior to binding the target to a probe and while the target biomolecule remains in the gel, (1) immersing the gel in a water miscible, aqueous extracting medium to shrink the gel by at least about ten percent and then (2) washing the gel with water to restore the gel to substantially its original size.

FIELD OF THE INVENTION

[0001] The present invention relates to the detection of targetsubstances and, more particularly, to detection of biomolecules directlyin a gel in which they have been separated by electrophoresis.

BACKGROUND OF THE INVENTION

[0002] Capture of target substances, such as biomolecules, on solidphases has been used for years in order to facilitate their subsequentidentification and to study their manner of interacting with othersubstances. Typical interactions are those between proteins, such asbetween antigens and antibodies, hormones and receptors, biotinylatedmolecules and biotin binding proteins, and the like. One method ofcapture is generally designated as “blotting,” whereby the targetsubstance is applied, directly or by transfer from another medium, to amembrane, such as nitrocellulose, polyvinyllidene difluoride (PVDF), ornylon.

[0003] Blotting is most frequently used in combination with known gelelectrophoresis procedures, whereby the target substance, e.g., anantigen, is first separated on a gel from other substances, typically bythe electrophoresis procedure commonly referred to as SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis). The targetsubstance is then transferred electrophoretically to a membrane.Subsequently, the target is associated, either directly or indirectlythrough an intermediate primary ligand, by an affinity interaction to areporter probe, such as an antibody, having a label attached theretowhich is capable of providing a detectable signal. Radioactive,calorimetric, fluorescent, or enzymatic labels are commonly employed toprovide the detectable signals which, in turn, indicate the presenceand/or location of the target molecule. Responses from radioactive andfluorescent labels are generally determined directly, while the response(luminescent, fluorescent, or colorimetric) elicited with enzymaticlabels is indirect in that the detectable signal results from the actionof the enzyme on an appropriate substrate.

[0004] Procedures for electrophoretic separation with subsequentblotting onto a membrane have historically been referred to in theliterature as either Western blotting, Northern blotting or Southernblotting. Western blotting refers to the identification of antigens astarget substances, while the latter two procedures refer toidentification of target RNA and DNA sequences, respectively. Morerecently, a variation of Western blotting, referred to as Far Westernblotting, has been used to characterize protein to protein interactions,other than antigen to antibody.

[0005] A drawback associated with Western type and other blottingtechniques is that they require time consuming and cumbersome steps.These include transferring the target substance from the gel to themembrane on which the substance is immobilized and then blocking themembrane. Some of these steps, particularly the transfer andimmobilization operations, may be detrimental to the protein beingassayed. For example, a change in antigenic nature of a protein mayprevent the corresponding antibody from binding and, therefore,detecting the target molecule. Also, the pattern obtained on a membranewhen a crude lysate is transferred may not be a true representation,since smaller molecular weight proteins transfer more efficiently thanlarger molecular weight proteins. Additionally, some proteins simply donot transfer well and, therefore, are not represented on the membrane atall.

[0006] Thus, “in gel” procedures, in which detection is accomplishedwithout removal of the target from the gel, have distinct advantages.And, while “in gel” techniques for detection of target molecules inpolyacrylamide gel were reported even before the advent of the abovedescribed blotting techniques (Burridge, K. (1976) Proc. Natl. Acad.Sci., USA, 73, 4457-4461; Rosta, J. A.; Kelly, P. T.; and Cotman, C. W.(1977) Anal. Biochem., 80, 336-376; and Olden, K. and Yamada K. M.(1977) Anal. Biochem, 78, 483-490), the procedures were very timeconsuming, entailing lengthy fixing, incubation and wash steps, whichgenerally took on the order of several days. Furthermore, “in gel”techniques have heretofore been considered not sufficiently sensitivefor the detection of target molecules using large reporter probes, e.g.,antigen-antibody interactions. The problem encountered is to obtainsufficient penetration of the probe or primary ligand into thethree-dimensional gel to achieve the required association of the targetand probe to permit detection at low concentrations of target.

[0007] Accordingly, a sought after objective is to provide an efficient“in gel” detection method which is sensitive and useful for thedetection of biomolecules. And, it is with respect to this objectivethat the present invention is directed.

SUMMARY OF THE INVENTION

[0008] In accordance with the present invention, there is provided animprovement to the method for detecting target biomolecules directly ina gel. The method to which the present improvement applies comprisesseparating a target biomolecule from other substances by polyacrylamidegel electrophoresis and, while the target biomolecule remains in thegel, associating it (either directly or indirectly) with a probe havinga label which is capable of providing a detectable signal. The methodfurther comprises removing any non-associated probe from the gel anddetecting the presence of the target biomolecule by observing a signalattributable to the presence of the labeled probe which has remainedassociated with the target.

[0009] The improvement provided by the present invention resides inaccomplishing the following two steps after the target molecule has beenseparated in the gel and prior to initiating the action(s) whereby thetarget biomolecule becomes associated with the probe. The first step isshrinking the gel by at least about ten percent (10%) and, preferably,at least about fifteen percent (15%). Then, in a second step, washingthe gel with water for a sufficient time to restore the gel tosubstantially its original size. After the foregoing procedures areaccomplished in accordance with this invention, the assay can becontinued with the incubation, washing, and detection steps asconventionally practiced.

[0010] As is apparent, once conventional electrophoresis has beencompleted, utilization of the improvement provided by the presentinvention permits rapid completion of the assay, since there is no needfor transfer to a membrane. The improved method provided by thisinvention can be accomplished in an elapsed time of about 30 minutesfrom the completion of electrophoresis to initial incubation with eitherprimary ligand or directly with target probe. This is to be comparedwith typical times of at least about 2.5-3 hours needed for membranepreparation, transfer, and blocking operations associated withconventional blotting procedures. And, since there is no necessity fortransferring or immobilizing the separated target molecule on amembrane, damage thereto is avoided. Quite surprisingly, the method ofthis invention is applicable for the direct detection in the gel ofsmall quantities of large molecules, such as proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the developed film that was exposed to the gel aftercompletion of the assay run in Example I.

[0012]FIG. 2A shows the developed film that was exposed to the gel aftercompletion of the assay run in Example II.

[0013]FIG. 2B shows the film that resulted from the conventional Westernblot procedure.

[0014]FIG. 3A shows the developed film that was exposed to the gel aftercompletion of the assay run in Example m and probed with anti-livingcolor primary antibody and detected with goat anti-rabbit antibody:HRP.

[0015]FIG. 3B shows the developed film that was exposed to the gel aftercompletion of the assay run in Example m and probed with anti-penta-Hisprimary antibody and detected with goat anti-mouse antibody:HRP.

[0016]FIG. 4 shows the image capture from a CCD camera of the gel aftercompletion of the assay run in Example IV.

DESCRIPTION OF THE INVENTION

[0017] The improvement provided by the present invention is generallyapplicable with respect to that technique known as SDS-PAGE, whereinsubstances are separated by electrophoresis in an aqueous polyacrylamidegel containing sodium dodecyl sulfate. It is particularly applicable forthe detection of proteinaceous biomolecules, such as antigens,antibodies, glycoproteins and other proteins. It also finds use withbiotinylated and other pre-labeled molecules separated by gelelectrophoresis. Once separated and identified on the gel, the proteinbands in the gel can be recovered from the gel for furthercharacterization and identification.

[0018] In view of the foregoing, the present invention provides animproved method of detecting a target biomolecule directly in apolyacrylamide gel. In a method comprising (1) separating a targetbiomolecule from other substances by electrophoresis in a gel, (2)associating the target biomolecule with a probe having a label which iscapable of providing a detectable signal, (3) removing anynon-associated probe from the gel, and (4) detecting the presence of thetarget biomolecule by observing a signal attributable to the presence ofthe labeled probe associated with the target biomolecule, theimprovement comprises (A) shrinking the gel by at least 10% and then (B)washing the gel with water for a sufficient time to restore the gel tosubstantially its original size prior to associating the targetbiomolecule with the probe and while the target biomolecule remainsseparated in the gel.

[0019] The invention is useful in connection with probes labeled with avariety of entities as previously identified which can directly orindirectly provide a detectable signal. Conventionally, the signal iscolorimetric, fluorescent or luminescent. The invention is, however,considered to be particularly useful in combination with probescontaining enzymatic labels which, on contact with a substrate, producea detectable luminescent or fluorescent signal. Horseradish peroxidase(HRP) and alkaline phosphatase are useful enzymes. When HRP is used,desirably the substrate is luminol.

[0020] A particularly preferred enzymatic detection system is describedin WO97/39142, published on Oct. 23, 1997, the disclosure of which ishereby incorporated by reference in its entirety. Therein, probescontaining as a label HRP are disclosed. In the presence of a luminolsubstrate, a source of hydrogen peroxide, and an enhancer, the HRPcatalyzes an oxidation reaction which yields an intense, long-lastingluminescent signal. With use of this system in combination with theimproved method of this invention, the detection,“in gel,” of proteinsusing large target biomolecules and primary ligands is enhanced. Systemsbased on this luminescent technology are marketed by Pierce ChemicalCompany, Rockford, Ill., under the Supersignal® trademark.

[0021] In practicing the present invention an important aspect isshrinking the gel after electrophoretic separation of the targetbiomolecule in the gel, but prior to initiating the incubation steps(directly with the labeled probe or indirectly first with a primaryligand) which result in achieving association of the target with theprobe. To this end, the wet gel can be immersed in a water miscible,aqueous extracting medium for a sufficient time to achieve the desiredshrinkage. Media comprised of water and a short-chain alcohol, such asmethanol, ethanol, isopropanol, or combinations thereof, are consideredmost useful. Other water miscible components, in lieu of or incombination with the alcohol, such as polyethylene glycol, can be usefulas well.

[0022] The alcohol in the aqueous extracting medium is present in anamount of about 10-75% v/v. Higher concentrations tend to shrink the gelexcessively, resulting in embrittlement of the gel, while, at lowerconcentrations, inadequate shrinkage is achieved in a reasonable amountof time. The concentration of alcohol is preferably about 40-60% v/vand, more preferably, about 50% v/v. Preferably, the alcohol ismethanol, ethanol, isopropanol or a combination thereof in an amount ofabout 35-65% v/v. More preferably, the alcohol is isopropanol in anamount of about 50% v/v. In further keeping with the present invention,the gel is maintained in the alcohol medium for a sufficient time toachieve a shrinkage of at least about 10%, based on the original surfacearea of the gel, preferably at least about 15%, and most preferablyabout 20%. Immersion for about 15 minutes is ordinarily adequate toachieve the required shrinkage. In order to minimize adverseembrittlement, shrinkage should be less than about 50%, and generallyless than about 35%.

[0023] After shrinking and still in keeping with the improvementprovided by this invention, the shrunk gel is washed with water, aloneor as an aqueous buffer, to remove the extracting medium from the geland, in turn, to re-hydrate the gel and restore it to substantially itsoriginal size, i.e., either the same or slightly (about 5%) smaller orlarger than its original size, generally slightly larger. While anymanner of washing can be employed, washing can be easily accomplishedusing a colander designed for gel detection procedures.

[0024] Once size restoration is accomplished by re-hydration throughwashing, the gel can then be directly incubated in a solution containingthe labeled target probe. Alternatively, if an indirect, sandwichapproach is utilized, incubation can first occur in a solutioncontaining a primary ligand having specific affinity for the targetbiomolecule followed by a second incubation in a solution containing areporter probe having affinity for the primary ligand. In eitherinstance, the probe becomes selectively associated with the targetbiomolecule. At some stage during the assay procedure,sensitivity/intensity of the assay can be enhanced by sonicating thegel. Subsequent to incubation(s), the assay can be completed followingconventional practice of washing and response elicitation.

[0025] Any one of many gels useful in electrophoresis applications, suchas SDS-polyacrylamide, which are either commercially available orindividually prepared are useful herein. The gels can be eitherhomogeneous or gradient. Pre-cast, Tris-Glycine, Bis-Tris, Tris-Acetateand Tricine gels commercially available from Novex have been found to beparticularly useful.

[0026] Furthermore, while the invention has been described withreference to one-dimensional electrophoresis, it is equally applicablewith respect to the electrophoresis operation accompanyingtwo-dimensional (2D) separations. In 2D separations, the step of gelelectrophoresis is preceded by a separation of substances by isoelectricfocusing. The 2D procedure is most useful when gel electrophoresis inthe first dimension alone is not sufficient to separate targetbiomolecules.

EXAMPLES

[0027] The following examples illustrates the present invention. Thereagents and materials used in the examples are as follows:

[0028] Pure glutathione-S-transferase (GST) and anti-GST, rabbitpolyclonal IgG were obtained from Santa Cruz BioTechnology (Santa Cruz,Calif.). E. coli bacterial cell lysate expressing recombinant mouseId-1:GST, mouse Id-2:GST and mouse Id-3:GST lystates were fromPharMingen (San Diego, Calif.) (for general discussion of Id proteins,see, e.g., Kadesch, T. (1993) Cell Growth Differ., 4, 49-55). Mouseanti-penta-His antibody and the His tag ladder were obtained from Qiagen(Chatsworth, Calif.); Living Color A.v. Peptide (specific for greenfluorescent protein [GFP]) antibody, rabbit, anti-6xHis antibody, HRPand GFP monoclonal antibody, mouse, were from ClonTech (Palo Alto,Calif.). Tris-Glycine SDS sample buffer (2×) was obtained from Novex(San Diego, Calif.). 9H-(1,3dichlor-9,9-dimethylacridin-2-one-7-yl)-phosphate (DDAO-phosphate) andgoat anti-rabbit, alkaline phosphatase were from Molecular Probes.Enhanced chemiluminescence (ECL) was obtained from Amersham PharmaciaBiotech. The Developer/Replenisher and the Fixer/Replenisher wereobtained from Sigma Chemical Corporation (St. Louis, Mo.). Gradient andhomogeneous Tris-Glycine polyacrylamide gels were obtained from Novex(San Diego, Calif.). The following materials were obtained from PierceChemical Company (Rockford, Ill.): HRP-labeled streptavidin; Y-Per®yeast protein extraction reagent; phosphate-buffered saline (PBS);Tris-buffered saline (TBS); Tween®-20; goat anti-rabbit, HRP; goatanti-mouse, HRP; electrophoresis buffer; bovine serum albumin (BSA);Supersignal® West Dura Extended Duration Substrate (stable peroxidebuffer and luminol enhancer); isopropanol; exposure film and magnesiumchloride. The yeast cells and lysates (GFP & GST) and the bacteriallysate (GFP/6xHis tagged) were prepared by conventional techniques.Mycobacterium Complex strain 101 (MAC 101) lysate and biotinylated humanrespiratory epithelial cell extract (B-Hep-2) were obtained from Dr.Venkata Reddy, School of Medicine at University of Illinois at Chicago,Rockford, Ill.

[0029] Gel sample preparation and SDS-PAGE electrophoresis wereaccomplished as follows. Pure protein samples were prepared by dilutionin the sample buffer such that the final protein concentration was 0.1-1ng/μl. For Example V, MAC 101 lysate was diluted so that the finalconcentration was from 0.5-1.5 μg/ml. Other lysate samples were diluted1:10-1:1,000 in 2× sample buffer. All of the samples were heated at 95°C. for 5 minutes and cooled before loading onto the gels (either 4-20%or 10-20% Tris-Glycine). The samples were separated by analyticalmini-gel SDS-PAGE, using Novex system, at 120 V until the dye frontreached halfway down, then at 180V until the dye front reached thebottom of the gel.

[0030] The following examples, Examples I-E, illustrate the presentinvention in an indirect assay protocol using a primary antibody (theprimary ligand) for initial bound association with the target proteinfollowed by incubation with a labeled secondary antibody (the reporterprobe), which binds to the primary antibody. Thus, the association oftarget and probe is indirect. Example IV illustrates the presentinvention in a direct assay protocol, in which the reporter probe isdirectly associated with the target.

Example I

[0031] The 4-20% Tris-Glycine gel (electrophoresed as described before)containing pure GST, mouse Id1:GST lysate, and yeast GST lysate wasincubated for 15 min in 50 ml of 50% isopropanol/H₂O to achieve about20% shrinkage and then washed with 100 ml H₂O (Milli-Q®) for 15 min torestore the gel to substantially its original size. The gel wasincubated with 20 ml rabbit anti-GST, diluted (1:1,000) in 1%BSA/PBS/0.05% Tween®-20, for 1 hr at room temperature (RT). The gel waswashed 3×10 min with 100 ml PBS/0.05% Tween®-20. An HRP-labeledsecondary antibody [goat anti-rabbit antibody/HRP (GAR/HRP)] stocksolution at 10 μg/ml was diluted 1:500 in 1% BSA/PBS/0.05% Tween®-20.The diluted antibody solution (20 ml) was added to the gel. The gel wasincubated for 1 hour at RT and then washed 3×10 min with 100 mlPBS/0.05% Tween®-20. The gel was incubated for 5 min at RT in 10 ml ofthe Supersignal® substrate working reagent. The gel was washed for 15sec with Milli-Q® Water. The gel was placed between cellophane sheetsand exposed to film for different lengths of time. The film wasdeveloped using the Developer/Replenisher and Fixer/Replenisher.

[0032]FIG. 1 is a copy of the developed film from the assay conductedaccording to Example I. Lanes 1, 2 and 3 correspond to 5, 10 and 1 ngpure GST, respectively. Lanes 4 and 5 correspond to GST lysate:Id-1(Santa Cruz Biotech) diluted 1:10 and 1:100, respectively. Lanes 6 and 7correspond to yeast GST lysate (Pierce) diluted 1:10 and 1:100,respectively. It is to be noted that a sensitive signal with lowbackground is obtained in this assay using GST and anti-GST, relativelylarge proteins.

Example II

[0033] Example I was repeated except that pure GFP/6xHis taggedproteins, GFP/6xHis expressed in bacterial cell lysate, and GFPexpressed in yeast lysate were used as targets and the primary antibodyused was the rabbit anti-living color antibody. The assay took about 3.5hrs to complete after electrophoresis. In addition, a conventionalWestern blot assay was run (taking about 6.5 hrs after electrophoresis)on the same target molecules, with detection being accomplished with anECL chemiluminescent substrate from Amersham Pharmacia Biochem.

[0034]FIG. 2 compares the “in gel” detection protocol according to thepresent invention (panel A) with the conventional Western Blot protocolusing ECL as a detection substrate (panel B). Lanes 1, 2 and 3correspond to 10, 5 and 1 ng pure GFP/6xHis-tagged, respectively. Lanes4 and 5 correspond to E. coli GFP/6xHis-tagged lysate diluted 1:100 and1:1,000, respectively. Lanes 6 and 7 correspond to yeast GFP lysatediluted 1:10 and 1:100, respectively. As can be seen the results arecomparable but, as noted above, the time needed to complete the assay inaccordance with the present invention was substantially shorter andinvolved less manipulative steps.

Example III

[0035] The target proteins run in Example II in accordance with thisinvention were repeated except that one gel (4-20%) was probed with thesame anti-living color primary antibody and detected with goat-antirabbit antibody:HRP (FIG. 3A) and another gel probed with anti-Penta-Hisprimary antibody (1:500 dilution) followed by goat-anti mouseantibody:HRP (1:250; 10 μg/ml) (FIG. 3B). Lanes 1, 2 and 3 correspond to10, 5 and 1 ng pure GFP/6xHis-tagged, respectively. Lanes 4 and 5correspond to E. coli GFP/6xHis-tagged lystate diluted 1:100 and1:1,000, respectively. Lanes 6 and 7 correspond to yeast GFP lystate1:10 and 1:100, respectively. This illustrates that detection using theinstant method is specific for the antigen being probed; a specificsignal is obtained in response to the primary antibody used in theassay. The Example shows that yeast GFP, which does not express a 6xHistag, is not detected by the anti-penta His antibody.

Example IV

[0036] The Tris-Glycine gel (10-20%), prepared as in Example II, withseparated GFP/6xHis pure protein, bacterial cell lysate expressingGFP/6xHis, and yeast lysate expressing GFP, was incubated for 15 minwith 50 ml of 50% isopropanol/H₂O and then washed with 100 ml H₂O(Milli-Q®) for 15 min. The gel was incubated in 20 ml HRP conjugatedprimary antibody (anti-6xHis;1:5,000), diluted in 1% BSA/PBS/0.05%Tween®-20, for 1 hr at RT. The gel was washed 3×10 min with 100 mlPBS/0.05% Tween®-20. The gel was then incubated for 5 min at RT in 10 mlSupersignal® Substrate working reagent. The gel was washed for 15 secwith Milli-Q® Water. The gel was placed between cellophane sheets andexposed to an Alpha Innotech CCD camera. As shown in FIG. 4, a directlylabeled primary antibody can be used in accordance with the presentinvention to detect antigens directly in the gel. Lanes 1, 2 and 3correspond to 10, 5 and 1 ng pure GFP/6xHis-tagged, respectively. Lanes4 and 5 correspond to E. coli GFP/6xHis-tagged lysate diluted 1:100 and1:1,000, respectively. Lanes 6 and 7 correspond to yeast GFP lysate 1:10and 1:100, respectively. Again, as in Example III, the antibody does notrecognize the yeast GFP which does not express the His tag.

[0037] Examples V and VI illustrate the invention where the target is aprotein in a cell lysate and the assay is indirect (Example V) or direct(Example VI).

Example V

[0038] An 8-16% gel was electrophoresed, as described previously,containing Mycobacterium avium complex lysate (MAC 101) diluted insample buffer at 15, 10 and 5 μg/lane. Duplicate samples of each lysatewere run on two different sides of the gel. The gel was incubated for 15min with 50 ml of 50% isopropanol/water and then washed with 100 ml(Milli-Q®) water for 15 min. The gel was cut in half through the middleso that each half of the gel contained the lysate samples. One of thegel halves was incubated in 20 ml biotinylated human respiratoryepithelial (B-Hep-2) cell extract at a starting concentration ofapproximately 0.5 mg/ml and then diluted 1:200 in 1×1% BSA/PBS/05%Tween®-20 for 1 hr at RT. The other half was incubated in just 1%BSA/PBS/05% Tween®-20 for 1 hr at RT. The gel halves were washed 3×10min with 100 ml PBS/05% Tween®-20. Both gel halves were then incubatedin 20 ml HRP-labeled Streptavidin solution, at 1 mg/ml, diluted 1:25,000in 1% BSA/PBS/05% Tween®-20, for 1 hr at RT. The gel halves were washed3×10 min with 100 ml PBS/05% Tween®-20. The gel halves were incubatedfor 5 min in 10 ml Supersignal® substrate working reagent and thenwashed with Milli-Q® water for 15 sec. The gel halves were placed inbetween cellophane and exposed to X-ray film for different lengths oftime. The film was developed using the Developer/Replenisher andFixer/Replenisher solutions.

[0039] A band corresponding to a 31 kD protein was observed in the gelincubated with B-Hep-2 extract. This was due to a reaction between aprotein in the B-Hep-2 extract and a protein in the MAC 101 lysate.Additional endogenous biotin bands were observed in the control gel,which was not incubated with the MAC 101 lysate, and in the gelincubated with the MAC 101 lysate.

Example VI

[0040] Yeast cells (1×10⁶) were washed 3×1 ml with PBS. The cells weredivided into 3 microcentrifuge tubes before the last wash. PBS (100 μl)was added to each tube after the last wash. The cells in two of thetubes were biotinylated with 5 μl of NHS-LC-Biotin (at 10 mg/ml in DMF)and Sulfo-NHS-LC-Biotin (at 10 mg/ml in Milli-Q water) respectively, for1 hr at 37° C. No biotinylation reagent was added to the third tube. Thecells were washed 2×1 ml with PBS, using a microcentrifuge at 10,000 rpmfor 5 min to separate the cells from the wash buffer. The cells werethen lysed with 100 μl of Y-PER® (Yeast Protein Extraction Reagent) for20 min at RT. The lysed cells were centrifuged for 5 min in amicrocentrifuge at 10,000 rpm to remove any debris and then mixed 1:3with 2× Novex Tris-Glycine SDS sample buffer and heated for 5 min at 95°C. Each sample (10 μl) was applied to a 4-20% gel. The gel waselectrophoresed as described previously. The gel was incubated for 15min with 50 ml of 50% isopropanol and then washed with 100 ml (Milli-Q®)water for 15 min. The gel was then incubated in 20 ml HRP-labeledStreptavidin solution, at 1 mg/ml, diluted 1:250,000 in 1% BSA/PBS/05%Tween®-20. The gel was washed 3×10 min with 100 ml PBS/05% Tween®-20.The gel was incubated for 5 min in 10 ml SuperSignal® substrate workingreagent and then washed with Milli-Q® water for 15 sec. The gel wasplaced in between cellophane and exposed to X-ray film for differentlengths of time. The film was developed using the Developer/Replenisherand Fixer/Replenisher solutions.

[0041] The results showed that different protein bands were observed forthe cells biotinylated with the NHS-LC-Biotin compared to thosebiotinylated with the water-soluble Sulfo-NHS-LC-Biotin. Thebiotinylation of the different proteins with these reagentsdistinguishes between integral membrane proteins, biotinylated with themembrane soluble NHS-LC-Biotin and the cell surface proteins,biotinylated with the water soluble, but membrane insoluble,Sulfo-NHS-LC-Biotin. These results show that it is possible todistinguish between cell surface proteins and integral membrane proteinsusing this technique. The control experiment did not show any proteinbands in response to the Streptavidin-HRP detection.

[0042] While the foregoing examples have employed the chemiluminescentsubstrate, luminol, and HRP, the following example, Example VII,illustrates the use of the present invention with the acridine basedfluorogenic substrate, DDAO-phosphate, together with alkalinephosphatase.

Example VII

[0043] The 10-20% Tris-Glycine gel electrophoresed as described inExample 1, containing pure GST and mouse Id1:GST, Id2:GST and Id3:GSTlysates, was incubated for 15 min in 50 ml of 50% isopropanol/H₂O andthen washed with 100 ml H₂O (Milli-Q®) for 15 min. The gel was incubatedin 20 ml rabbit anti-GST, diluted (1:1000) in 1% BSA/TBS/0.05%Tween®-20, for 1 hr at RT. The gel was washed 3×10 min with 100 mlTBS/0.05% Tween®-20. An alkaline phosphatase labeled secondary antibody(goat anti-rabbit antibody/alkaline phosphatase stock solution at 2mg/ml) was diluted 1:1,000 in 1% BSA/TBS/0.05% Tween®-20. The dilutedantibody solution (20 ml) was added to the gel. The gel was incubatedfor 1 hr at RT and then washed 2×10 min with 100 ml TBS/0.05% Tween®-20and 1×10 min with 10 ml TBS (pH 9.5) containing 1 mM MgCl₂. The gel wasincubated for 15 min at RT in 10 ml DDAO-phosphate substrate workingsolution [DDAO-phosphate was reconstituted at 1.25 mg/ml and thendiluted 1:1,000 in TBS (pH 9.5) containing 1 mM MgCl₂]. The substratewas removed and the gel was scanned using a Typhoon instrument fromAmersham Pharmacia Biotech. The instrument was set at 646 nm absorbancemaxima and 659 emission maxima.

[0044] All the samples, pure GST and GST lysates, were detected withthis method. Thus, the example illustrates the feasibility of theinvention with fluorescent substrates.

[0045] While the foregoing examples have illustrated use of the presentinvention in connection with interactions between proteins, it can alsobe extended to study the interaction of other biomolecules, such asnucleic acids. Here, the invention finds particular use in connectionwith assays in which interactions are detected by retardation phenomena.For example, the method can be used in the detection of DNA and RNAinteractions with each other or with proteins. Such assays are practicedby a procedure in which the biomolecules of interest are incubatedtogether in solution and then separated by gel electrophoresis.Retardation assays are based on the principle that the free biomolecule,e.g., DNA, will migrate faster than a complex, e.g., of DNA and protein.By detecting a difference in band migration in the gel the existence ofan interaction can be established.

[0046] All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

[0047] Preferred embodiments of this invention are described herein,including the best mode known to the inventors for carrying out theinvention. Variations of those preferred embodiments may become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

What is claimed is:
 1. In a method for detecting a target biomoleculedirectly in a polyacrylamide gel comprising (1) separating the targetbiomolecule from other substances by electrophoresis in the gel and,while the target molecule remains in the gel, (2) associating the targetbiomolecule with a probe having a label which is capable of providing adetectable signal, (3) removing any non-associated probe from the gel,and (4) detecting the presence of the target biomolecule by observing asignal attributable to the presence of the labeled probe associated withthe target biomolecule; the improvement wherein, prior to associatingthe target biomolecule with the probe and while the target biomoleculeremains separated in the gel, (A) shrinking the gel by at least about10% and then (B) washing the gel with water for a sufficient time torestore the gel to substantially its original size.
 2. The method ofclaim 1 wherein shrinkage of the gel in (A) is accomplished by immersingthe gel in a water miscible, aqueous extracting medium for a sufficienttime to shrink the gel by at least about 10% and less than about 50%. 3.The method of claim. 2 wherein shrinkage of the gel is at least about15% and less than about 35%.
 4. The method of claim 3 wherein theaqueous extracting medium comprises water and a short chain alcoholpresent in an amount of about 10-75% v/v.
 5. The method of claim 4wherein the alcohol is methanol, ethanol, isopropanol, or combinationsthereof present in an amount of about 35-65% v/v.
 6. The method of claim5 wherein the alcohol is isopropanol present in an amount of about 50%v/v.
 7. The method of claim 5 wherein shrinkage of the gel is about 20%.8. The method of claim 6 wherein shrinkage of the gel is about 20%. 9.The method of claim 1 wherein the signal is fluorescent or luminescent.10. The method of claim 9 wherein the label is an enzyme and thefluorescent or luminescent signal detected is generated through actionof the enzyme on a substrate.
 11. The method of claim 10 wherein theenzyme is alkaline phophatase and the signal observed is fluorescent.12. The method of claim 10 wherein the enzyme is horseradish peroxidaseand the signal observed is luminescent.
 13. The method of claim 12wherein the substrate is luminol.